PDJ amplicon in triple negative breast cancer

Amplification of chromosome 9p24.1 targeting PD-L1, PD-L2, and JAK2 (PDJ amplicon) is present in subsets of triple negative breast cancers (TNBCs) and is associated with poor clinical outcomes. However, the prevalence of PDJ+ TNBCs varies extensively across studies applying different methods for interrogating samples of interest. To rigorously assess the prevalence of PDJ amplicons in TNBC, its prognostic value and whether it is enriched by chemotherapy, we interrogated 360 TNBC samples including 74 surgical resections from patients treated in the neoadjuvant setting, and tissue microarrays (TMAs) with 31 cases from African American women and 255 resected non-metastatic cases, with a 3 color fluorescence in situ hybridization (FISH) assay targeting the 9p24.1 PDJ amplicon, 9q24.3, and 9q34.1. Samples with mean PDJ signal of > 4.5 copies, and ratios of PDJ/9q24 ≥ 2 and/or PDJ/9q34.1 ≥ 2 were called amplified (PDJ+). Correlative analyses included the association of tumor infiltrating lymphocytes (TILs) with PDJ amplicons in TNBCs. In addition, we investigated intratumor copy number of PDJ amplicons in PDJ+ and PDJ− TNBCs. Matched pre- and post-neoadjuvant treatment biopsies were available from patients (n = 6) to evaluate the effects of therapy on PDJ status. Our study provides a rigorous analysis of the prevalence, distribution, and clinical correlatives of the PDJ amplicon in TNBC.

www.nature.com/scientificreports/ (IHC) assays provide the basis for established clinical tests to screen samples for PD-1/PD-L1 activation. However, these assays have variable thresholds with heterogeneous scoring on tumor and immune cells. In contrast, genomic amplifications are specific to tumor cells and provide robust therapy markers (e.g., HER2, FGFR1/2, EGFR), that can be objectively scored in slide-based assays. The PD-L1 and PD-L2 genes localize to 9p24.1 adjacent to JAK2. We and others have identified a 9p24.1 amplicon with the shortest region of overlap including JAK2 and PD-L1 (PDJ amplicon) in a variety of tumors [17][18][19][20][21][22][23][24][25][26][27][28] . High-level 9p24.1 amplification correlates with activation of the JAK/STAT pathway [29][30][31] . Overexpression of both JAK2 and PD-L1 mRNA has been associated with 9p24.1 amplification in a JAK2-dependent manner 29,30 . The JAK2/STAT3 signaling pathway is required for growth of CD44(+) CD24(−) stem cell-like breast cancer cells, particularly in basal-like breast cancers and is independently associated with breast cancer metastasis and poor prognosis 32,33 . Notably our study of flow sorted tumor populations confirmed that the PDJ amplicon is present in chemoradiation naïve resected cases, includes JAK2 and PD-L1, and correlates with increased RNA expression of JAK2 (p < 0.0001) and of PD-L1 (p < 0.0229) 29 . In addition, our preclinical studies have shown that PD-L1 expression is markedly and rapidly inducible by low-dose IFN-γ in a PDJ amplicon copy-number dependent manner, mimicking an in situ inflammatory response 34 . Thus, rather than constitutive overexpression, the PDJ amplicon is associated with a dynamic cytokine-inducible PD-L1 expression on tumor cells. The prevalence of the PDJ amplicon was found to be ≥ tenfold higher in TNBCs relative to other solid tumors 31 . Notably, the presence of PDJ amplicons has been reported to be limited in newly diagnosed TNBC but enriched by neoadjuvant chemotherapy 30 . These reports highlight a need for additional studies in independent patient cohorts, including pre-and post-neoadjuvant treatment samples to investigate the clinical significance of PDJ amplification in TNBC.
The highly aberrant nature of TNBC genomes and the enriched presence of 9p24.1 amplicons make TNBC a highly favorable tumor model to investigate clinical and genomic correlates of the PDJ amplicon 35 . In order to determine the prevalence of PDJ amplification and its association with clinical variables we developed a robust three color clinical FISH assay that can be applied to routinely collected pathology slides and tissue microarrays (TMAs). In this study we interrogated 360 TNBC cases from three distinct cohorts, an African American cohort (n = 31) from Howard University, surgical resections (n = 74) including 6 cases with matching pre and post neoadjuvant chemotherapy biopsies from Mayo Clinic Arizona (MCA); and treatment naive resected cases (n = 255) from a TMA provided by the Mayo Clinic Breast Cancer Specialized Program in Research Excellence (SPORE). Correlative analyses of the Mayo Clinic TMA results evaluated the association of histopathology and tumor infiltrating lymphocytes (TILs) in those TNBCs with PDJ amplicons. Our study provides a rigorous validation of the prevalence of PDJ amplicons in TNBC with a robust FISH assay across 3 independent cohorts, and a unique analysis of clinical correlates for this subset of TNBCs.
The distribution of PDJ amplicon copies per cell in the TNBC biopsies was found to vary significantly with increasing mean copy number status (Fig. 3). Tissues with PDJ deletions (x̅ < 2) or neutral copy numbers (x̅ = 2) tended to exhibit narrow ranges (0-3 copies) of PDJ amplicon copies per cell. In contrast, tumors with PDJ copy number gains or amplifications were found to have broader distributions that included individual cells with 10 or more copies in the same sample. The nine positive cases included cells with as many as 26 copies and up to 17/50 (34%) cells with ≥ 10 copies in each PDJ + sample (Fig. 2C,D). Images from the FISH analyses were examined to assess the spatial distribution of PDJ amplicons in TNBC tissues with increased copy numbers (Figs. 1, 2, 3). The PDJ + TNBCs displayed remarkable heterogeneity with cells possessing ≥ 15-20 PDJ copies located immediately adjacent to tumor cells with only 1-2 copies. In addition to the PDJ + biopsies, three PDJ gain samples (2 post and 1 pre neoadjuvant biopsies) had at least one cell with ≥ 10 copies of PDJ amplicon.
In the African American (AA) cohort, ages ranged from 28 to 76 (average 47), and stage at diagnosis ranged from I-IV. We obtained results from a single TMA slide that passed quality control including a series of diploid normal samples. Scoring was targeted to 20 intact cells per case. Six of 31 (19%) tumors on the slide had PDJ amplification by FISH, of which five were from breast tissue and one was from a lymph node (Fig. 1E www.nature.com/scientificreports/ The third cohort was a TMA containing surgically treated stage I-III TNBCs (Table 3) 36 . We obtained FISH data from 255 cases on the TMA including 143 cases with replicate spots that could be read and 112 with results from a single spot on the array. Thirty-six of 255 (14.1%) evaluable cases were PDJ + based on our scoring criteria ( Table 2). Similar to our cohort of resected cases the distribution of PDJ + cells within each positive case included cells with as many as 30 copies and up to 28/50 (56%) cells with ≥ 10 copies in each PDJ + sample (Fig. 1B,C). Correlative analyses of results from the Mayo Clinic Breast Cancer SPORE TMA identified an association of increased TILs in those TNBCs with PDJ amplicons (Wilcoxon Rank Sum 2-sided p-value = 0.01) ( Table 4). The median stromal TIL counts for the PDJ positive and PDJ negative groups were 30 and 20.0, respectively. PDJ amplicon and chemotherapy. There were seven patients from our neoadjuvant cohort with matching pre-and post-treatment biopsies. FISH results were obtained from six of the matching pairs that included variable baseline PDJ copy numbers (Table 5). In none of these cases did we observe an enrichment of the PDJ amplicon in the post treatment sample. Rather the PDJ copy number was stable in 5/6 of these cases and decreased in one amplified case.

Discussion
TNBC is an aggressive disease with poor clinical outcomes. Thus, there is a need to identify markers that can be exploited for improved clinical care. We and subsequently others have identified the PDJ amplicon as a clinically relevant driver of aggressive TNBC. Prior studies assessing resections of primary TNBC tissues have associated PDJ amplification with increased tumor size, frequent lymph node involvement and advanced disease (Stages 3-4), and reduced progression free survival (PFS) and overall survival (OS) 29,30 . Furthermore, there is emerging data suggesting that the presence of the amplicon is associated with increased JAK2/pSTA3 signaling and a reprogrammed immune environment. Immune checkpoint blockade with anti-PD-L1 therapies that overcome tumor-mediated local immunosuppression have been shown to induce regression in 13-38% of metastatic TNBCs that are PD-L1 + by immunohistochemistry (IHC) staining [29][30][31] . Response to ICIs in TNBCs has been associated with tumor PD-L1 expression 37 . However, the significant temporal and spatial heterogeneity of PD-L1 www.nature.com/scientificreports/ expression seen with IHC complicates its use as a robust biomarker to identify TNBC patients likely to respond to ICI therapies [38][39][40] .
Genomics-based biomarkers of PD-L1 expression may provide alternative or complementary means of addressing tumor heterogeneity and identifying cohorts of TNBC patients likely to respond to ICI therapies. The PDJ amplicon is associated with tumor PD-L1 expression in a dynamic, copy-number dependent manner and is regulated by active JAK-STAT signaling 32,34,41 . Our preclinical studies have shown that PD-L1 expression is markedly and rapidly inducible by low-dose IFN-γ in a PDJ amplicon copy-number dependent manner, mimicking an in situ inflammatory response. Thus, rather than constitutive overexpression, the PDJ amplicon is associated with a dynamic cytokine-inducible PD-L1 expression on tumor cells that may correlate with response to PD-L1 inhibition. Notably, our previous studies have shown that PD-L1 protein expression is highly variable within PDJ + tumors with a striking difference in staining intensity between the undifferentiated regions of the tumor (IHC score 5) and those that were differentiated (IHC score 0) 42 . Of significant interest in future studies will be to assess whether PDJ amplifications as measured with our FISH assay, can predict response to ICI therapies regardless of the level of PD-L1 expression measured by IHC 41,43,44 .
Our neoadjuvant TNBC cohort consists of 97 biopsies from 74 patients from a single institution (MCA). The median age of patients in this cohort was 59 with an enrichment for smaller (74.3% T1-T2) and earlier stage (72.9% 1-2) tumors. Notably, this cohort included six cases with matching pre-and post-treatment biopsies. The PDJ amplicon was noted to have broader distributions with 1-20 PDJ copies per single tumor cell in PDJamplified patients, which is consistent with the high degree of heterogeneity seen with PD-L1 expression in TNBCs. Strikingly, three PDJ gain patient samples were observed to have at least one cell with ≥ 10 PDJ copies. The clinical significance of these PDJ + cells in otherwise PDJ-tumors remains to be determined.
TNBC disproportionately affects young African American women 45 . In addition, AA women with TNBC have worse clinical outcomes than women of European descent. However, it remains to be determined whether this is due to distinct molecular features of disease or to socioeconomic factors including disparities in access to health-care treatment, co-morbid disease, and income. Although limited in the number of evaluable cases, the prevalence of PDJ + cases in the AA cohort was similar to that from the surgical resection cohort and the Mayo www.nature.com/scientificreports/ Clinic Breast Cancer SPORE TMA of untreated TNBCs. Larger follow up studies with additional cases from AA women will provide a more complete profile of PDJ amplicons in this population. The presence of TILs has been reported in patients who responded to ICIs in several clinical trials. For example, in IMpassion130, joint analysis of PD-L1 and TILs found that cases which were PD-L1 positive (SP142) and had TILs > 10% had outcomes which appeared superior to cases which were PD-L1 positive alone as per the primary analysis 46 . Our results from the Mayo Clinic Breast Cancer SPORE TMA identified an association of increased stromal TILs in those TNBCs (14%) with PDJ amplicons targeting PD-L1 and JAK2 (P < 0.01).
The limitations of this study listed include the lack of detailed genomic characterization of the patient samples and post-surgical clinical outcomes ( Table 6). The analysis of matching pre and post neoadjuvant treated samples was limited to 6 pairs. In addition, our cohorts do not contain biopsies from patients treated with ICIs. However, our survey of three distinct cohorts of TNBC cases establishes a baseline prevalence (14%) for PDJ + in this aggressive subtype, includes scoring of single cells within each tumor, and provides preliminary correlative association with increased TILs, a signature associated with improved responses to ICI therapies.
We validated our FISH assay with aCGH results from a subset of flow sorted TNBCs in the surgical cohort (Supplemental Fig. 1). Furthermore, our 3 probe design enabled the discrimination of PDJ amplifications from whole chromosome 9 aneuploidies that are prevalent in TNBC. The height of this amplicon in our studies includes aCGH log 2 ratios > 4 corresponding to FISH copy numbers > 20 consistent with amplification of genomic

PDJ amplification and immune cells in TNBC. Our results from the Mayo Clinic Breast Cancer SPORE
TMA identified an association of increased stromal TILs in those TNBCs (14%) with PDJ amplicons targeting PD-L1 and JAK2 (P < 0.01). The median stromal TIL counts for the PDJ positive and PDJ negative groups were 30 and 20.0, respectively. Notably the presence of TILs has been reported in patients who responded to ICIs in several clinical trials. For example, in IMpassion130, joint analysis of PD-L1 and TILs found that cases which were PD-L1 positive (SP142) and had TILs > 10% had outcomes which appeared superior to cases which were PD-L1 positive alone as per the primary analysis. TILs alone also predicted benefit from the addition of atezolizumab. Our hypothesis is the PDJ amplicon is a robust genomic marker for those TNBC patients likely to respond to ICI. Given direct associations of PDJ + with PD-L1 and stromal TILs, our initial focus will be patients treated with a PD-L1 inhibitor (atezolizumab). However, we are aware of the use and clinical benefit of PD-1 inhibitors (pembrolizumab) in early stage and metastatic TNBC so evaluation will extend to these patient populations in future studies.

Evidence for a clinical impact of 9p24.1 amplification in other tumor types. In lymphoma,
JAK2 has been shown to up regulate the transcription of both PD-1 ligands (PD-L1, PD-L2) while increasing sensitivity to JAK2 inhibitors in a dose dependent manner 25 . In addition, there have been case reports of significant responses to immune checkpoint inhibition in solid tumors with 9p24.1 amplicons targeting JAK2, PD-L1 and PD-L2 [47][48][49] . Notably these occurred in advanced microsatellite stable tumors including non-small cell lung cancer, a colon adenocarcinoma, and a cancer of unknown primary origin. There is also emerging data that a 9p24.1 amplicon is present in Epstein-Barr virus (EBV)-positive gastric cancers and in squamous cell carcinoma (SCC) of the cervix or vulva 25,27,50 . In our preliminary published data, we found the PDJ amplicon in a subset (3-5%) of glioblastomas (2/44) and colorectal cancers (2/68) 29 . Similar findings have been reported in these and other solid tissue tumors 26,43,51 . Although relatively rare the presence of PDJ amplicons in common tumor types such as lung and colorectal cancers highlight the potential clinical impact of this genomic lesion in addition to TNBC.

Materials and methods
Clinical samples. All patients gave informed consent for collection and use of the samples. All tumor samples were histopathologically evaluated prior to genomic analysis. All research was performed in accordance with relevant guidelines/regulations and conformed to the Helsinki Declaration (https:// www. wma. net/ polic iespost/ wma-decla ration-of-helsi nki-ethic al-princ iples-for-medic al-resea rch-invol ving-human-subje cts/). www.nature.com/scientificreports/

Fluorescence in situ hybridization (FISH).
Three color assay. The clinical samples and the TMAs were interrogated with a multicolor FISH assay consisting of three chromosome 9 probes that simultaneously target JAK2 and PD-L1 (Tamra), chromosome 9 peri-centromere (SpectrumGreen), and 9q34.1 (TexasRed) regions ( Supplementary Fig. 1). Each probe consisted of two non-overlapping bacterial artificial chromosome (BAC) clones that mapped to the three loci of interest. This enables discrimination of PDJ amplicons from chromosome 9 ploidies that can occur in the background of genomic instability in aneuploid cancer genomes. The enumeration probe set was applied to individual slides, hybridized, and washed according to published protocols 56 57 . Specifically, PDJ amplification was called in samples with an average copy-number > 4.5, a ratio of PDJ to 9q24.3 (peri-centromere) and/or PDJ to 9q34.1 ≥ 2.0, and at least one cell